A Fungal N‐Dimethylallyltryptophan Metabolite from Fusarium fujikuroi

The range of secondary metabolites (SMs) produced by the rice pathogen Fusarium fujikuroi is quite broad. Several polyketides, nonribosomal peptides and terpenes have been identified. However, no products of dimethylallyltryptophan synthases (DMATSs) have been elucidated, although two putative DMATS genes are present in the F. fujikuroi genome. In this study, the in vivo product derived from one of the DMATSs (DMATS1, FFUJ_09179) was identified with the help of the software MZmine 2. Detailed structure elucidation showed that this metabolite is a reversely N‐prenylated tryptophan with a rare form of prenylation. Further identified products probably resulted from side reactions of DMATS1. The genes adjacent to DMATS1 were analyzed; this showed no influence on the biosynthesis of the product.     

Kinome-Wide Profiling Identifies Human WNK3 as a Target of Cajanin Stilbene Acid from Cajanus cajan (L.) Millsp.

Pigeon Pea (Cajanus cajan (L.) Millsp.) is a common food crop used in many parts of the world for nutritional purposes. One of its chemical constituents is cajanin stilbene acid (CSA), which exerts anticancer activity in vitro and in vivo. In an effort to identify molecular targets of CSA, we performed a kinome-wide approach based on the measurement of the enzymatic activities of 252 human kinases. The serine-threonine kinase WNK3 (also known as protein kinase lysine-deficient 3) was identified as the most promising target of CSA with the strongest enzymatic activity inhibition in vitro and the highest binding affinity in molecular docking in silico. The lowest binding affinity and the predicted binding constant pKi of CSA (−9.65 kcal/mol and 0.084 µM) were comparable or even better than those of the known WNK3 inhibitor PP-121 (−9.42 kcal/mol and 0.123 µM). The statistically significant association between WNK3 mRNA expression and cellular responsiveness to several clinically established anticancer drugs in a panel of 60 tumor cell lines and the prognostic value of WNK3 mRNA expression in sarcoma biopsies for the survival time of 230 patients can be taken as clues that CSA-based inhibition of WNK3 may improve treatment outcomes of cancer patients and that CSA may serve as a valuable supplement to the currently used combination therapy protocols in oncology.     

Identification of Biologically Diverse Tetrahydronaphthalen-2-ols through the Synthesis and Phenotypic Profiling of Chemically Diverse,Estradiol-Inspired Compounds

Combining natural product fragments to design new scaffolds with unprecedented bioactivity is a powerful strategy for the discovery of tool compounds and potential therapeutics. However, the choice of fragments to couple and the biological screens to employ remain open questions in the field. By choosing a primary fragment containing the A/B ring system of estradiol and fusing it to nine different secondary fragments, we were able to identify compounds that modulated four different phenotypes: inhibition of autophagy and osteoblast differentiation, as well as potassium channel and tubulin modulation. The latter two were uncovered by using unbiased morphological profiling with a cell-painting assay. The number of hits and variety in bioactivity discovered validates the use of recombining natural product fragments coupled to phenotypic screening for the rapid identification of biologically diverse compounds.     

Identification of the Target Protein of Agelasine D,a Marine Sponge Diterpene Alkaloid,as an Anti‐dormant Mycobacterial Substance

One of the major reasons for the wide epidemicity of tuberculosis and for the necessity for extensive chemotherapeutic regimens is that the causative agent, Mycobacterium tuberculosis, has an ability to become dormant. Therefore, new lead compounds that are anti‐bacterial against M. tuberculosis in both active and dormant states are urgently needed. Marine sponge diterpene alkaloids, agelasines B, C, and D, from an Indonesian marine sponge of the genus Agelas were rediscovered as anti‐dormant‐mycobacterial substances. Based on the concept that the transformants over‐expressing targets of antimicrobial substances confer drug resistance, strains resistant to agelasine D were screened from Mycobacterium smegmatis transformed with a genomic DNA library of Mycobacterium bovis BCG. Sequence analysis of the cosmids isolated from resistant transformants revealed that the responsible gene was located in the genome region between 3475.051 and 3502.901 kb. Further analysis of the transformants over‐expressing the individual gene contained in this region indicated that BCG3185c (possibly a dioxygenase) might be a target of the molecule. Moreover, agelasine D was found to bind directly to recombinant BCG3185c protein (K_D 2.42 μm), based on surface plasmon resonance (SPR). This evidence strongly suggests that the BCG3185c protein is the major target of agelasine D, and that the latter is the anti‐mycobacterial substance against dormant bacilli.     

Dual-PKS Cluster for Biosynthesis of a Light-Induced Secondary Metabolite Found from Genome Sequencing of Hyphodiscus hymeniophilus Fungus

Filamentous fungi are known producers of important secondary metabolites. In spite of this, the majority of these organisms have not been studied at the genome level, leaving many of the bioactive molecules they produce undiscovered. In this study, we explore the secondary metabolite potential of an understudied fungus, Hyphodiscus hymeniophilus. By sequencing and assembling the first genome from this genus, we show that this fungus has genes for at least 20 natural products and that many of these products are likely novel. One of these metabolites is identified: a new, red-pigmented member of the azaphilone class, hyphodiscorubrin. We show that this metabolite is only produced when the fungus is grown in the light. Furthermore, the biosynthetic gene cluster of hyphodiscorubrin is identified though homology to other known azaphilone producing clusters.     

Heterologous Biosynthesis of Fungal Indole Sesquiterpene Sespendole

Indole sesquiterpene sespendole, which has been isolated from the filamentous fungus Pseudobotrytis terrestris FKA‐25, is a specific inhibitor of lipid droplet synthesis in mouse macrophages. The biosynthetic pathway that involves genes encoding six enzymes (spdEMBQHJ) was elucidated through heterologous expression of spd genes in Aspergillus oryzae, biotransformation experiments, and in vitro enzymatic reactions with a recombinant protein, thereby revealing the mechanism underlying the characteristic modification on the indole ring, catalyzed by a set of prenyltransferase (SpdE)/cytochrome P450 (SpdJ) enzymes. Functional analysis of the homologous genes encoding these enzymes involved in the biosynthesis of lolitrem allowed a biosynthetic pathway for the bicyclic ring skeleton fused to the indole ring to be proposed.     

Multimodal Molecular Imaging and Identification of Bacterial Toxins Causing Mushroom Soft Rot and Cavity Disease

Soft rot disease of edible mushrooms leads to rapid degeneration of fungal tissue and thus severely affects farming productivity worldwide. The bacterial mushroom pathogen Burkholderia gladioli pv. agaricicola has been identified as the cause. Yet, little is known about the molecular basis of the infection, the spatial distribution and the biological role of antifungal agents and toxins involved in this infectious disease. We combine genome mining, metabolic profiling, MALDI-Imaging and UV Raman spectroscopy, to detect, identify and visualize a complex of chemical mediators and toxins produced by the pathogen during the infection process, including toxoflavin, caryoynencin, and sinapigladioside. Furthermore, targeted gene knockouts and in vitro assays link antifungal agents to prevalent symptoms of soft rot, mushroom browning, and impaired mycelium growth. Comparisons of related pathogenic, mutualistic and environmental Burkholderia spp. indicate that the arsenal of antifungal agents may have paved the way for ancestral bacteria to colonize niches where frequent, antagonistic interactions with fungi occur. Our findings not only demonstrate the power of label-free, in vivo detection of polyyne virulence factors by Raman imaging, but may also inspire new approaches to disease control.     

EBDCs杀菌剂及代谢物ETU的毒理学研究进展

乙撑双二硫代氨基甲酸酯(EBDCs)是一类非内吸性杀菌剂,这类杀菌剂如代森锰锌、代森锰等,广泛用于马铃薯、谷物、苹果、梨和叶菜作物。概述了EBDCs类杀菌剂在环境中的移动、残留、降解、代谢、代谢物,评价了其急性毒性、慢性作用、致癌风险和毒理学研究进展。     

Total Synthesis and Structural Revision of Kasumigamide,and Identification of a New Analogue

Kasumigamide is an antialgal hybrid peptide–polyketide isolated from the freshwater cyanobacterium Microcystis aeruginosa (NIES-87). The biosynthetic gene cluster was identified from not only the cyanobacterium but also Candidatus “Entotheonella”, associated with the Japanese marine sponge Discodermia calyx. Therefore, kasumigamide is considered to play a key role in microbial ecology, regardless of the terrestrial and marine habitats. We now report synthetic studies on this intriguing natural product that have led to a structural revision and the first total synthesis. During this study, a new analogue, deoxykasumigamide, was also isolated and structurally validated. This study confirmed the presence of the unusual pathway in the biosynthesis of a hybrid peptide–polyketide natural product.     

Inspired by Nature: The 3‐Halo‐4,5‐dihydroisoxazole Moiety as a Novel Molecular Warhead for the Design of Covalent Inhibitors

Over the past few decades, there has been an increasing interest in the development of covalent enzyme inhibitors. As it was recently re‐emphasized, the selective, covalent binding of a drug to the desired target can increase efficiency and lower the inhibitor concentration required to achieve a therapeutic effect. In this context, the naturally occurring antibiotic acivicin, and in particular its 3‐chloro‐4,5‐dihydroisoxazole scaffold, has provided a wealth of inspiration to medicinal chemists and chemical biologists alike. In this Concept, to underline the great potentiality that the 3‐halo‐4,5‐dihydroisoxazole warhead has in drug discovery, we present a number of examples, grouped by their potential biological activity and targets, in which this scaffold has been fruitfully used to develop novel biologically active compounds. Through these examples, we show that the 3‐halo‐4,5‐dihydroisoxazole moiety represents an outstanding warhead with high potential for the design of novel covalent enzyme inhibitors.     

Identification of Pyrroloformamide as a Cytokinesis Modulator

Discovered in the late 1940s, the pyrrolinonodithioles represent a family of potent disulfide‐containing natural products. Although they are understood in a synthetic and biosynthetic context, the biological role of these materials remains unresolved. To date, their activity has been suggested to arise through regulating RNA metabolism, and more recently they have been suggested to function as backup thiols for detoxification. Using materials identified through a natural products program, we now identify the biological function of one member of this family, pyrroloformamide, as an antimitotic agent acting, in part, by disrupting cytokinesis.     

Understanding Substrate Selectivity of Phoslactomycin Polyketide Synthase by Using Reconstituted in Vitro Systems

Polyketide synthases (PKSs) use simple extender units to synthesize complex natural products. A fundamental question is how different extender units are site-specifically incorporated into the growing polyketide. Here we established phoslactomycin (Pn) PKS, which incorporates malonyl- and ethylmalonyl-CoA, as an in vitro model to study substrate specificity. We combined up to six Pn PKS modules with different termination sites for the controlled release of tetra-, penta- and hexaketides, and challenged these systems with up to seven different extender units in competitive assays to test for the specificity of Pn modules. While malonyl-CoA modules of Pn PKS exclusively accept their natural substrate, the ethylmalonyl-CoA module PnC tolerates different α-substituted derivatives, but discriminates against malonyl-CoA. We show that the ratio of extender transacylation to hydrolysis controls incorporation in PnC, thus explaining site-specific selectivity and promiscuity in the natural context of Pn PKS.     

The Identification of a Novel Natural Activator of p300 Histone Acetyltranferase Provides New Insights into the Modulation Mechanism of this Enzyme

Many severe human pathologies are related to alterations of the fine balance between histone acetylation and deacetylation; because not all such diseases involve hypoacetylation, but also hyperacetylation, compounds able to enhance or repress the activities of histone acetyltransferases (HATs) could be promising therapeutic agents. We evaluated in vitro and in cell the ability of eleven natural polyisoprenylated benzophenone derivatives to modulate the HAT activity of p300/CBP, an enzyme that plays a pivotal role in a variety of cellular processes. Some of the tested compounds bound efficiently to the p300/CBP protein: in particular, guttiferone A, guttiferone E and clusianone inhibit its HAT activity, whereas nemorosone showed a surprising ability to activate the enzyme. The ability of nemorosone to penetrate cell membranes and modulate histone acetylation into the cell together with its high affinity for the p300/CBP enzyme made this compound a suitable lead for the design of optimized anticancer drugs. Besides, the studies performed at a cellular and molecular level on both the inhibitors and the activator provided new insights into the modulation mechanism of p300/CBP by small molecules.     

The Evolution and Impact of Total Synthesis on Chemistry,Biology and Medicine

The advent of organic synthesis in the nineteenth century sparked a revolution in chemistry that led from a serendipitous discovery to the art and science that it is today. Its creative nature turned into enabling technologies that set in motion entire new industries such as the dye and pharmaceutical enterprises and helped elucidate and confirm structures of countless natural products. It also served as the locomotive for discovery and invention of new reactions, synthetic strategies and technologies, and delivered myriad valuable compounds, more or less complex, for research and applications in our everyday lives. Today it is a partner of biology in the quest of understanding living nature and applying the gathered intelligence to discover and develop newer and more effective drugs.     

Targeted Discovery of Tetrapeptides and Cyclic Polyketide-Peptide Hybrids from a Fungal Antagonist of Farming Termites

Herein, we report the targeted isolation and characterization of four linear nonribosomally synthesized tetrapeptides (pseudoxylaramide A–D) and two cyclic nonribosomal peptide synthetase-polyketide synthase-derived natural products (xylacremolide A and B) from the termite-associated stowaway fungus Pseudoxylaria sp. X187. The fungal strain was prioritized for further metabolic analysis based on its taxonomical position and morphological and bioassay data. Metabolic data were dereplicated based on high-resolution tandem mass spectrometry data and global molecular networking analysis. The structure of all six new natural products was elucidated based on a combination of 1D and 2D NMR analysis, Marfey's analysis and X-ray crystallography.     

Automated Identification of Depsipeptide Natural Products by an Informatic Search Algorithm

Nonribosomal depsipeptides are a class of potent microbial natural products, which include several clinically approved pharmaceutical agents. Genome sequencing has revealed a large number of uninvestigated natural‐product biosynthetic gene clusters. However, while novel informatic search methods to access these gene clusters have been developed to identify peptide natural products, depsipeptide detection has proven challenging. Herein, we present an improved version of our informatic search algorithm for natural products (iSNAP), which facilitates the detection of known and genetically predicted depsipeptides in complex microbial culture extracts. We validated this technology by identifying several depsipeptides from novel producers, and located a large number of novel depsipeptide gene clusters for future study. This approach highlights the value of chemoinformatic search methods for the discovery of genetically encoded metabolites by targeting specific areas of chemical space.